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EP3748009A1 - Synthèse de l'acétate de (r)-pantolactone - Google Patents

Synthèse de l'acétate de (r)-pantolactone Download PDF

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Publication number
EP3748009A1
EP3748009A1 EP19178973.4A EP19178973A EP3748009A1 EP 3748009 A1 EP3748009 A1 EP 3748009A1 EP 19178973 A EP19178973 A EP 19178973A EP 3748009 A1 EP3748009 A1 EP 3748009A1
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EP
European Patent Office
Prior art keywords
pantolactone
enzyme
acetate
enzymes
group
Prior art date
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EP19178973.4A
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German (de)
English (en)
Inventor
Myria Bekerle-Bogner
Jonathan Alan Medlock
Georg STEINKELLNER
Gernot Strohmeier
Kerstin Steiner
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DSM IP Assets BV
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DSM IP Assets BV
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Priority to EP19178973.4A priority Critical patent/EP3748009A1/fr
Priority to CN202010511527.6A priority patent/CN112048537A/zh
Publication of EP3748009A1 publication Critical patent/EP3748009A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/001—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by metabolizing one of the enantiomers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/26—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D307/30—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/32—Oxygen atoms
    • C07D307/33—Oxygen atoms in position 2, the oxygen atom being in its keto or unsubstituted enol form
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/004—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of alcohol- or thiol groups in the enantiomers or the inverse reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01074—Cutinase (3.1.1.74)

Definitions

  • the present invention relates to a 2-step process comprising step (1) kinetic resolution of R/S-pantolactone using an enantioselective acetylating enzyme and step (2) racemization of (S)-pantolactone in the presence of at least one specific chemical catalyst leading to highly enriched (R)-pantolactone acetate.
  • step (1) and (2) might be carried-out in a one-pot reaction/system.
  • Pantothenate or vitamin B5 is a member of the B complex of vitamins which is essential for mammals including humans. It has to be supplied as food or feed additive. In cells, pantothenate is used primarily for the biosynthesis of coenzyme A and acyl carrier protein. These essential coenzymes function in the metabolism of acyl moieties, which form thioesters with the sulfhydryl group of the 4'-phosphopantethein portion of these molecules. Importantly, only (R)-pantothenate is biologically active as vitamin.
  • Pantothenate has been synthesized conventionally via chemical synthesis from bulk chemicals.
  • the substrates required for chemical synthesis are expensive and the racemic intermediates have to be optically resolved.
  • Bacterial or microbial systems, if available, that produce enzymes useful in pantothenate biosynthesis processes, are not yet feasible for industrial production because of insufficient yield and/or selectivity of the used enzymes.
  • the present invention is directed to (1) a process for conversion of racemic (R/S)-pantolactone via catalytic action of enzymes having carboxylic ester hydrolase [EC 3.1.1] activity, including triacylglycerol lipase enzymes [EC 3.1.1.3] and cutinase enzymes [EC 3.1.1.74], wherein (R/S)-pantolactone and an acetate ester are selectively converted into (R)-pantolactone-acetate via enantioselective acetylation under non-aqueous conditions.
  • enzymes having carboxylic ester hydrolase [EC 3.1.1] activity including triacylglycerol lipase enzymes [EC 3.1.1.3] and cutinase enzymes [EC 3.1.1.74]
  • (R/S)-pantolactone and an acetate ester are selectively converted into (R)-pantolactone-acetate via enantioselective acetylation under non-aqueous conditions.
  • any enzyme with activity of ⁇ / ⁇ -hydrolases particularly enzymes of class [EC 3.1.1], isolated from various sources can be used, particularly such enzymes isolated from fungi including yeast, bacteria or higher eukaryotes.
  • the enzyme is selected from the class [EC 3.1.1.3] or [EC 3.1.1.74], including but not limited to Candida antarctica lipase A (CalA) including CalA-like enzymes isolated from Kurtzmanomyces sp.
  • NCBI accession code 20429169 Malassezia furfur (NCBI accession code 73765555) or Ustilago maydis (NCBI accession code 71018653), cutinase 1 (Cut1) or other lipases, such as e.g. isolated from Thermomyces lanuginosus (Humicola lanuginosa), Fusarium solani, Humicola insolens, Candida antarctica including C. antarctica lipase B (CalB), or Rhizomucor miehei.
  • the enantioselective enzyme to be used in step (1) and as defined herein is a lipase, such as e.g. selected from Fusarium solani, preferably F. solani pisi cutinase 1 (Cut1), more preferably a polypeptide with at least about 80%, such as e.g. 85, 90, 95, 98 or 100% identity to SEQ ID NO:1, including polynucleotides encoding said polypeptide such as e.g. the polynucleotide of SEQ ID NO:2. Cut1 is commercially available from various suppliers, e.g. such as ASA Spezialenzyme GmbH (Germany).
  • ASA Spezialenzyme GmbH Germany
  • the enantioselective enzyme to be used in step (1) and as defined herein is selected from Candida, preferably Candida antarctica lipase B (CalB), more preferably a polypeptide with at least about 80%, such as e.g. 85, 90, 95, 98 or 100% identity to SEQ ID NO:3, including polynucleotides encoding said polypeptide such as e.g. the polynucleotide of SEQ ID NO:4.
  • CalB is commercially available from various suppliers, e.g. such as Novozymes or Merck.
  • the enantioselective enzyme to be used in step (1) and as defined herein is a lipase, such as e.g. selected from Thermomyces lanuginosus (Humicola lanuginosa), preferably Thermomyces lanuginosus (Humicola lanuginosa) lipase (TLL), more preferably a polypeptide with at least about 80%, such as e.g. 85, 90, 95, 98 or 100% identity to SEQ ID NO:5, including polynucleotides encoding said polypeptide such as e.g. the polynucleotide of SEQ ID NO:6.
  • TLL is commercially available from various suppliers, e.g. such as Novozymes or Merck.
  • ⁇ / ⁇ -hydrolase lipase
  • lipase or "enzyme having lipase activity”
  • pantolactone hydrolyzing enzyme acetylating enzyme
  • alcoholysis enzyme alcoholysis enzyme
  • hydrolysis enzyme acetylating enzyme
  • alcoholysis enzyme alcoholysis enzyme
  • hydrolysis enzyme enantioselective enzyme
  • (R)-selective enzyme or “(R/S)-pantolactone transesterase”
  • It refers to an enzyme having lipase or ⁇ / ⁇ -hydrolase activity which is involved in conversion, i.e.
  • Suitable acetate esters to be used for acetylation of (R/S)-pantolactone are selected from either unsaturated, saturated or aromatic acetates including but not limited to vinyl acetate, phenylvinyl acetate, ethoxyvinyl acetate, isoprenyl acetate, isopropenyl acetate, ethyl acetate or p-chlorophenyl acetate, preferably vinyl acetate.
  • the enzymatic conversion of (R/S)-pantolactone and an acetate ester as defined herein into (R)-pantolactone acetate, i.e. enantioselective hydrolysis/acetylation, as described herein can be performed with isolated enzymes, such as e.g. the acetylating enzymes as defined herein, particularly lipases or cutinases, such as e.g. CalB, Cut1, TLL, that might be expressed in a suitable host cell, such as e.g. yeast or bacteria, preferably Pichia or Escherichia, such as e.g. as heterologous enzymes.
  • isolated enzymes such as e.g. the acetylating enzymes as defined herein, particularly lipases or cutinases, such as e.g. CalB, Cut1, TLL, that might be expressed in a suitable host cell, such as e.g. yeast or bacteria, preferably Pichia or Escherichia, such
  • the enantioselective enzymes as described herein are heterologous expressed in Pichia pastoris. Isolation of suitable enzymes can be done by known methods, the respective enzymes might be further used in isolated form or in the form of a cell extract, powder or liquid formulations, i.e. in immobilized or non-immobilized form.
  • the enzymes - either modified or non-modified - as defined herein, such as e.g. CalB, Cut1, TLL, that are used in a process for enantioselective acetylation of (R/S)-pantolactone and an acetate ester as defined herein, are used in isolated form, such as e.g.
  • liquid or immobilized form such as either covalently bound or adsorbed, e.g. on epoxy carrier, depending on the supplier.
  • immobilized forms the enzymes might be used several times with more or less the same performance (so-called recycling).
  • the described enzyme forms apply to both the modified and non-modified enzymes.
  • step (1) conversion, “hydrolysis”, “alcoholysis”, “acetylation”, “enantioselective acetylation”, “enantioselective hydrolysis” or “enantioselective alcoholysis” in connection with step (1), i.e. enzymatic catalysis of the enzymes [EC 3.1.1] as described herein, preferably enzymes selected from class [EC 3.1.1.3] or [EC 3.1.1.74], leading to enantioselective production of (R)-pantolactone acetate from racemic pantolactone and an acetate ester as defined herein, are used interchangeably herein.
  • conversion rate refers to the activity of the ⁇ / ⁇ -hydrolases, particularly CalB, Cut1, TLL, which can be measured as conversion of (R/S)-pantolactone into pantolactone acetate.
  • E value refers to the general enantioselectivity of the enzyme independent of the conversion rate.
  • enantiomeric excess ee as used herein refers to the degree to which a sample contains one enantiomer (such as e.g. (R)-pantolactone) in higher amount than the other enantiomer (such as e.g. (S)-pantolactone), see Helpfile on selectivity, Uni Graz, 2012: http://biocatalysis.uni-graz.at/enantio/DataFiles/Selectivity-Help.pdf.
  • racemic (R/S)-pantolactone refers to a mixture comprising (R) and (S)-pantolactone in any ratio, including a mixture wherein the (R) and (S)-enantiomers are present in equal quantities, i.e. a so-called racemate.
  • the term "specific activity” or "activity” with regards to enzymes means its catalytic activity, i.e. its ability to catalyze formation of a product from a given substrate.
  • the specific activity defines the amount of substrate consumed and/or product produced in a given time period and per defined amount of protein at a defined temperature.
  • specific activity is expressed in ⁇ mol substrate consumed or product formed per min per mg of protein.
  • An enzyme is active, if it performs its catalytic activity in vivo, i.e.
  • the present invention is directed to a process according to step (1) for enantioselective hydrolysis of (R/S)-pantolactone, wherein the (R/S)-pantolactone and an acetate ester as defined herein is enzymatically converted into pantolactone acetate and (S)-pantolactone with a conversion rate in the range of at least about 20, such as e.g. at least about 25, 30, 35, 40, 45, 50, 55, or up to 60 % conversion of racemic pantolactone into pantolactone acetate.
  • the pantolactone acetate is present as (R)-enantiomer, with preferred E values of at least about 10, such as e.g. 15, 20, 25, 30, 40, 50, 60 or even 100 and more.
  • the process according to the present invention comprising step (1) and step (2) leads to a conversion of (R/S)-pantolactone and an acetate ester as defined herein into (R)-pantolactone acetate with a conversion rate of at least about 20, such as e.g. at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or even up to 100% and E values of at least about 10, such as e.g. 15, 20, 25, 30, 40, 50, 60 or even 100 and more.
  • one or more amino acid modification(s) might be introduced, preferably into the substrate binding region of the enzymes.
  • modified cutinase enzymes including but not limited to Cut1, preferably Cut1 from Fusarium solani pisi. These results might be applicable to other Cut1 enzymes from other organisms or respective lipases as defined herein.
  • the present invention is directed to a hydrolase as defined herein that has been modified via the introduction of one or more amino acid substitution(s) in a sequence corresponding to amino acid residues 28 to 169 in the polypeptide with at least about 80% identity to SEQ ID NO:1, such as e.g. substitution(s) of one or more amino acid(s) in a sequence corresponding to residues T28 to L169 in the polypeptide according SEQ ID NO:1, more particularly via introduction of one or more amino acid substitution(s) at position(s) corresponding to residue(s) selected from the group consisting of position 28, 66, 169, and combinations thereof in the polypeptide with at least about 80%, such as e.g.
  • a "modified” enzyme e.g. modified lipase or cutinase
  • Preferred enzyme is Cut1, such as Cut1 from Fusarium solani pisi.
  • a "non-modified” enzyme as defined herein, particularly Cut1, as used herein refers to the respective endogenous or commercially available enzymes not carrying one or more amino acid substitution(s) as defined herein.
  • a host cell carrying/expressing a modified catalytic activity as defined herein, particularly Cut1, comprising one or more amino acid substitution(s) as defined herein is referred to as "modified” host cell.
  • the respective host cell carrying a non-modified catalytic activity, such as encoding the wild-type Cut1 gene, is referred to as “non-modified” host cell.
  • the modified enzyme as defined herein in particular enzyme with modified Cut1 activity, i.e. improved enantioselective activity towards (R)-pantolactone acetate, comprises an amino acid substitution at a position corresponding to residue 28 in the polypeptide according to SEQ ID NO:1, preferably substitution of threonine by lysine or alanine (T28L or T28A).
  • E values in the range of about 27 (with toluene as co-solvent) to about 42 (with EtOAc as co-solvent) after 18h at 40°C of reaction could be achieved, compared to step (1) using the respective non-modified enzyme, including but not limited to Cut1 according to e.g. SEQ ID NO:1, with E values of about 23 (with toluene as co-solvent) to about 33 (with methyl tert-butylether as co-solvent) after 18h at 40°C.
  • the modified enzyme as defined herein in particular enzyme with modified Cut1 activity, i.e. improved enantioselective activity towards (R)-pantolactone acetate, comprises an amino acid substitution at a position corresponding to residue 66 in the polypeptide according to SEQ ID NO:1, preferably substitution of lysine by alanine (L66A).
  • E values in the range of at least about 25 (with toluene as co-solvent) to about 38 (with EtOAc as co-solvent) after 18h at 40°C of reaction could be achieved, compared to step (1) using the respective non-modified enzyme, including but not limited to Cut1 according to e.g. SEQ ID NO:1, with E values of at least about 23 (with toluene as co-solvent) to about 33 (with MTBE as co-solvent) after 18h at 40°C.
  • the modified enzyme as defined herein in particular enzyme with modified Cut1 activity, i.e. improved enantioselective activity towards (R)-pantolactone acetate, comprises an amino acid substitution at a position corresponding to residue 169 in the polypeptide according to SEQ ID NO:1, preferably substitution of valine by alanine (V169A).
  • E values in the range of at least about 27 (with toluene as co-solvent) to about 38 (with methyl tert-butylether as co-solvent) after 18h at 40°C of reaction could be achieved, compared to step (1) using the respective non-modified enzyme, including but not limited to Cut1 according to e.g. SEQ ID NO:1, with E values of at least about 23 (with toluene as co-solvent) to about 33 (with methyl-tert-butylether as co-solvent) after 18h at 40°C.
  • the described amino acid substitution(s) at a position corresponding to residues as disclosed herein might be combined with each other, leading to conversion rates and/or an E value which are increased as compared to single mutations, such as e.g. an increase in the range of at least about 2, 5, 10, 20, 50% compared to single mutations.
  • Selectivity mutations in enzyme active sites often have an additive or preferably synergistic effect when they are combined, thus increasing the overall selectivity of the enzyme.
  • combination of the selectivity mutations described herein might improve the E value of the enzymes even more, such as e.g. leading to E values of about 10 or more and/or conversion rates in the range of about 50% or more.
  • mutagenesis may be performed in different ways, such as for instance by random or site-directed mutagenesis, physical damage caused by agents such as for instance radiation, chemical treatment, or insertion of a genetic element.
  • agents such as for instance radiation, chemical treatment, or insertion of a genetic element.
  • the skilled person knows how to introduce mutations.
  • a host cell comprising and expressing a modified or non-modified enantioselective enzyme as defined herein, such as e.g. modified or non-modified Cut1 activity, i.e. improved enantioselective activity towards (R)-pantolactone acetate, is used in a process for production of (R)-pantolactone acetate from conversion of racemic (R/S)-pantolactone and an acetate ester as defined herein.
  • the host cell comprising the modified/non-modified enzyme may be cultured in an aqueous medium supplemented with appropriate nutrients under aerobic or anaerobic conditions and as known by the skilled person for the respective host cells.
  • such cultivation is in the presence of proteins and/or co-factors involved in transfer of electrons, as known in the art.
  • the cultivation/growth of the host cell may be conducted in batch, fed-batch, semi-continuous or continuous mode.
  • (R)-pantolactone acetate might be isolated and/or optionally further purified from the mix of (R)-pantolactone acetate and (S)-pantolactone.
  • step (1) as described herein is carried out using an immobilized modified or non-modified enzyme for enantioselective acetylation of (R/S)-pantolactone with an acetate ester to mainly form (R)-pantolactone acetate.
  • an immobilized modified or non-modified enzyme for enantioselective acetylation of (R/S)-pantolactone with an acetate ester to mainly form (R)-pantolactone acetate Such process might be performed under conditions known in the art, e.g. such as in an aqueous medium.
  • said process according to step (1) is performed under conditions which are not interfering with the racemization of (S)-pantolactone of step (2).
  • suitable solvents which could be used in step (1) include but are not limited to aliphatic hydrocarbons, aromatic hydrocarbons, chlorinated hydrocarbons, esters, ethers, and/or alcohols, particularly under conditions comprising at least about 1% of water. Immobilization might be performed with Lifetech ECR8285 (Purolite® Life Sciences) or other methods know to the skilled person.
  • the amount of enzyme used according to step (1) might vary, such as in the range of at least about 2, 5, 6, 8, 10, 12, 15, 18, 20 mg enzyme per mL solvent (dry immobilized enzyme in mass%), such as e.g. 10 to 17 mg/mmol pantolactone, with reactions carried out at about 35-45°C, such as e.g. about 40°C.
  • organisms such as e.g. microorganisms, fungi, algae or plants also include synonyms or basonyms of such species having the same physiological properties, as defined by the International Code of Nomenclature of Prokaryotes or the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code).
  • the present invention is directed to a 2-step process, wherein (R/S)-pantolactone and an acetate ester are selectively converted into (R)-pantolactone-acetate via enantioselective acetylation and as described above as step (1) and wherein the by-product (S)-pantolactone is converted back into (R/S)-pantolactone in the presence of one or more specific transition metal complex(es) as (chemical) catalyst(s) and as described herein as step (2).
  • conversion in connection with step (2) refers to the activity of the transition metal complex used for selective racemization of (S)-pantolactone into (R/S)-pantolactone.
  • racemization is art-recognized and refers to conversion of either (R)- or (S)-enantiomers in a racemic form of (R/S)-isomers. As used herein, it refers to the selective conversion of (S)-pantolactone into (R/S)-pantolactone.
  • the present invention is directed to a process comprising step (1) and step (2), wherein both steps are performed in one reaction, i.e. in one-pot. They might be alternatively performed in a loop or cascade reactor.
  • step (1) and (2) are performed in a loop or cascade reactor
  • the temperature in step (1) and (2) might vary, e.g. with step (1) being carried out at about 40°C and step (2) being carried out at higher temperature, e.g. at least about 50°C.
  • the reaction time might vary, with durations of about 18 to 72h or up to 258h are possible.
  • step (2) the racemization of (S)-pantolactone according to step (2) is performed under conditions which are not interfering with the enzymatic acetylation of step (1) and vice-versa.
  • the racemization of step (2) and as described herein is carried out in the presence of at least one specific transition metal complex as (chemical) catalyst, wherein the transition metal(s) is/are selected from the group consisting of Ru, Rh, Ir, Fe, Mn and Co, preferably selected from Ru or Ir.
  • the at least one transition metal complex might comprise one or more ligands.
  • a catalyst to be used in step (2) and as defined herein is the "Shvo catalyst", which is 1-hydroxytetraphenyl-cyclopentadienyl(tetraphenyl-2,4-cyclopentadien-1-one)- ⁇ -hydrotetracarbonyldiruthenium(II). This catalyst was first described by Shvo et al. (J.Am.Chem.Soc., 1986, 108, pp. 7400-7402 ).
  • a suitable solvent for the racemization as of step (2) and as described herein might be selected from aliphatic hydrocarbons, aromatic hydrocarbons, chlorinated hydrocarbons, esters, ethers, and alcohols, preferably selected from THF, 2-methyl-THF, toluene, xylene, cyclohexane, or ethyl acetate.
  • Solvents to be used in a one-pot reaction as described herein wherein step (1) is performed in the presence of immobilized enzymes might be selected from organic solvents as described above, such as e.g. toluene, dichloromethane, acetone, methyl tert-butylether (MTBE), ethyl acetate and the like.
  • step (2) and as described herein might be carried out at elevated temperatures, such as e.g. temperatures up to 100°C, such as e.g. between about 25 to 100°C, preferably between about 40 to 90°C.
  • the temperature might be selected from about 25 to 80°C, preferably from about 30 to 70°C, such as 40°C, but particularly preferred at least about 50°C.
  • step (1) and (2) as described herein is used for production of (R)-pantolactone acetate.
  • the (R)-pantolactone acetate is furthermore hydrolyzed to (R)-pantolactone and converted to pantothenic acid and its derivatives by well-known transformations.
  • Example 1 General methods, media, strains and plasmids
  • Cut1, CalB or TLL variants i.e. modified Cut1, CalB or TLL were codon-harmonized and cloned into the methanol inducible Pichia pastoris expression vector pAaHswa ( Ahmad et al., Appl Microbiol Biotechnol (2014) 98:5301-5317 ).
  • Chiral GC-FID-measurements were done on an Agilent HP 6890 gas chromatograph equipped with hot-split injector, autosampler and FID detector. Column used was Chiralsil-Dex 25 m x 0.32 mm, 0.25 ⁇ m film (9082223, #CP7503, Varian). The inlet temperature was 200°C, detector temperature was 220°C, with 1.0 ml/min constant N2 flow, split ratio 10:1, 110°C hold 10 min, 10°C/min to 160°C hold 2 min. Retention times are listed below: Table 1. Retention times (t ret ) as measured according to the method described in the text.
  • SEQ ID NO:1 and 3 show the mature polypeptide, whereas SEQ ID NO:5 still includes the signal sequence (underlined).
  • (S)-Pantolactone can be obtained from the mixture obtained in step (1) (see Ex. 3) by separation from (R)-pantolactone acetate.
  • the mixture of (S)-pantolactone and (R)-pantolactone acetate from step (1) (see Ex. 3) can be used directly.
  • Exp. means “experiment”, with Exp.1a given as comparative example.
  • Examples 3 and 4 can be combined together and carried out in a one-pot reaction, in a loop or cascade reactor with step (1) and (2) taking place at different sections (areas) of the loop.

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Citations (2)

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Publication number Priority date Publication date Assignee Title
US2688027A (en) * 1952-04-22 1954-08-31 Nopco Chem Co Racemization of optical isomers
EP0443406A2 (fr) * 1990-02-17 1991-08-28 Hoechst Aktiengesellschaft Procédé de séparation enzymatique des formes racémiques de pantolactone

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MXPA02003417A (es) * 1999-10-29 2002-08-20 Basf Ag L-pantolactona-hidrolasa y un procedimiento para la obtencion de d-pantolactona.
CN1768141B (zh) * 2003-03-28 2012-10-10 帝斯曼知识产权资产管理有限公司 泛酸内酯水解酶
CN105308181A (zh) * 2013-06-21 2016-02-03 帝斯曼知识产权资产管理有限公司 低pH的HNL突变体

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2688027A (en) * 1952-04-22 1954-08-31 Nopco Chem Co Racemization of optical isomers
EP0443406A2 (fr) * 1990-02-17 1991-08-28 Hoechst Aktiengesellschaft Procédé de séparation enzymatique des formes racémiques de pantolactone

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Title
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AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1998, WILEY
DATABASE UniProt [online] 8 May 2019 (2019-05-08), SOLIDAY, C.L. ET AL.: "Full=Cutinase 1, EC=3.1.1.74, Full=Cutin hydrolase 1", XP002796553, Database accession no. P00590 *
GAMALEVICH, G.D. & SEREBRYAKOV, E.P.: "Chemistry of Natural Compounds and Bioorganic Chemistry, Enantioselectivity of enzymatic acylation of some structurally various racemic alcohols in anhydrous aprotic media", RUSSIAN CHEMICAL BULLETIN, vol. 46, no. 1, January 1997 (1997-01-01), pages 171 - 183, XP002796552 *
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SHVO ET AL., J.AM.CHEM.SOC., vol. 108, 1986, pages 7400 - 7402

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